KR20190015869A - A method for analyzing eml4-alk gene variance - Google Patents

Abstract

According to an embodiment of the present invention, provided is a primer for qRT-PCR which can detect an EML4-ALK gene mutation based on blood circulating cancer cells at a limit of detection more sensitive than a conventional method. According to another embodiment of the present invention, the EML4-ALK gene mutation can be detected using blood circulating lung cancer cells even in patients with lung cancer, in which an ALK-FISH test cannot be performed due to difficulty in collecting solid biopsies of lung cancer tissues, thereby providing a screening method for lung cancer patients to see whether an anticancer agent targeting the EML4-ALK gene mutation can be applied to the lung cancer patients.

Description

A METHOD FOR ANALYZING EML4-ALK GENE VARIANCE [0002]

The present invention relates to a method of detecting EML4-ALK mutation, and more particularly, to a PCR primer pair for detecting EML4-ALK mutation based on circulating cancer cells, a method for detecting EML4-ALK mutation using nested PCR, To a method of screening non-small cell lung cancer patients against cancer drugs using cancer cells.

Lung cancer is the fourth most common cancer among Koreans in 2010 (10.3%) among cancer patients in Korea (202,053). However, the 5 - year survival rate is 19.7%, which is worse than other cancers. Lung cancer is classified into non - small cell lung cancer and small cell lung cancer according to the size and shape of cancer cells microscopically. Thus, distinguishing between non - small cell lung cancer and small cell lung cancer is different from clinical course and treatment. According to the data released by the Central Cancer Registry in 2011, there were 192,561 cases of cancer in Korea in 2009, of which 14,300 cases of non-small cell lung cancer occurred in men and women, and 19,685 cases of non-small cell lung cancer occurred in 72,672 cases Respectively. The male to female sex ratio was 2.5: 1, which was higher in males (published by the Central Cancer Center of the Ministry of Health and Welfare on December 29, 2011). ALK-positive non-small cell lung cancer is a lung cancer caused by the fusion of two genes, ALK and EML4, with a mutation in the EML4-ALK gene corresponding to 4 to 7% (7% of Asians) of all lung cancer patients and 167 Ten of the non-small cell lung cancer patients in Korea were reported to have the EML4-ALK mutation (J. Korean Med. Sci., 27: 228-230, 2012).

The targeted anticancer drug for non-small cell lung cancer by the EML4-ALK mutation is XALKORI (crizotinib) developed by Pfizer. Since the approval of Abbott's companion diagnostics in collaboration with XALKORI by the Food and Drug Administration (FDA) in August 2011, it has become a widely used, clinically proven and standardized method of genetic testing in the American Oncology community. It has been approved for CE-IVD testing in Europe and has been used in medical practice since September 2011 and is mainly used to support academic research and evaluation of new therapies. After the emergence of XALKORI, the treatment of non-small cell lung cancer (EGFR, EML4-ALK) was followed by the Iressa or conventional cytotoxic chemotherapy (with low therapeutic efficacy and high side effects) with or without mutation after EGFR mutation test. The paradigm shifts in the way of choosing the best cure / conventional cytotoxic chemotherapy. The ALK gene mutation shows various types of gene fusion. Immunohistochemistry, RT-PCR, and FISH can be used for clinical tests. Currently, the standard diagnostic test for crizotinib is FDA . However, this method is difficult and complicated and has a problem of costly and time-consuming to test a large number of patients. In addition, it is known to reach anatomical characteristics (Lung Cancer 84: 39-44, 2014, Cancer Cytopathol. Epub ahead of print 2014.4.10), and an alternative search is urgently required.

On the other hand, CTC (circulating tumor cell) is a small number of tumor cells that move away from the primary tumor tissue and circulate in the bloodstream. There are one to several thousand blood circulating cancer cells per 1 mL of blood depending on the cancer type. The use of blood-circulating cancer cells is a powerful technology that can replace conventional biopsy. It is a powerful technology that can reduce the patient's pain and risk due to low cost, biopsy and quick selection of target drug to prolong patient's life .

Therefore, the present inventors have made intensive efforts to develop a gene testing method for enabling an anticancer drug, for example, crizotinib, to be administered to patients who are difficult to collect lung cancer tissue, and as a result, To thereby confirm the EML4-ALK mutation of the circulating lung cancer cells with high accuracy, thereby completing the present invention.

It is an object of the present invention to provide a pair of PCR primers for detection of EML4-ALK mutation for highly accurate confirmation of the blood circulating cancer cell-based EML4-ALK gene mutation. It is another object of the present invention to provide a method for detecting V1 type mutation of EML4-ALK with high accuracy. It is still another object of the present invention to provide a method for detecting V3 type mutation of EML4-ALK with high accuracy.

It is another object of the present invention to provide a method for screening cancer patients through the use of blood-derived cancer cells in patients with non-small cell lung cancer, through the availability of anticancer drugs to cancer patients.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are not intended to limit the invention to the precise form disclosed. There will be.

According to an aspect of the present invention, there is provided a method for assaying mutation in EML4-ALK gene,

Obtaining a liquid biopsy sample from a cancer patient;

Separating blood circulating cancer cells from the liquid biopsy sample using the biochip;

Separating the RNA from the isolated blood circulating cancer cells;

Performing qRT-PCR using the separated RNA as a template and using two qRT-PCR primers;

Performing nested PCR using the product from the qRT-PCR as a template and using two nested primers for the two qRT-PCR primers; And

Detecting a mutation type of the EML-ALK gene as a product from the nested PCR;

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In an embodiment of the present invention, the liquid biopsy sample may be blood.

In an embodiment of the present invention, the cancer may be lung cancer.

In an embodiment of the present invention, the cancer may be non-small cell lung cancer.

In an embodiment of the present invention, the step of separating the blood circulating cancer cells may be performed under an atmospheric pressure of 1000 to 1020 hPa.

In an embodiment of the present invention, the biochip may be a high-density microchip coated with a BSA solution.

In an embodiment of the present invention, the high density microchip may have size specificity.

In an embodiment of the present invention, the BSA solution coating may be coated at a concentration of 0.05 to 0.15%.

In an embodiment of the present invention, the mutation type of the EML4-ALK gene may be a V1 type or a V3 type.

In an embodiment of the present invention, one of the two primers for qRT-PCR may be a primer for forward qRT-PCR and the other may be a primer for reverse qRT-PCR.

In an embodiment of the present invention, the forward qRT-PCR primer may be one selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 4.

In an embodiment of the present invention, the reverse qRT-PCR primer may be SEQ ID NO: 2.

In an embodiment of the present invention, one of the two nested primers may be a forward nested primer and the other may be a reverse nested primer.

In one embodiment of the present invention, the forward nested primer may be one selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 10.

In an embodiment of the present invention, the reverse nested primer may be SEQ ID NO: 2 or SEQ ID NO:

According to another aspect of the present invention, there is provided a cancer patient screening method comprising:

The gene mutation of the cancer patient obtained through the EML4-ALK gene mutation analysis method according to the present invention

(i) exons 1-13 of the EML4 gene and exons 20-29 of the ALK gene;

(ii) exons 1-20 of the EML4 gene; exons 20-29 of the ALK gene;

(iii) exons 1-6a of the EML4 gene and exons 20-29 of the ALK gene;

(iv) exons 20-29 of the exon 1-6b + ALK gene of the EML4 gene;

(v) exon 15 of the EML4 gene and exon 20-29 of the ALK gene;

(vi) a linker consisting of an exon 14 + 11bp-sized oligonucleotide of the EML4 gene and an exon 20-29 of the ALK gene;

(vii) exon 2 of the EML4 gene + exon 20-29 of the ALK gene; And

(viii) an exon 2 of the EML4 gene, an intron 19 of the ALK gene, and an exon 20-29 of the ALK gene, to indicate that the anticancer agent is effective for the cancer patient.

In an embodiment of the present invention, the cancer may be lung cancer.

In an embodiment of the present invention, the cancer may be non-small cell lung cancer.

In an embodiment of the present invention, the anticancer agent may be chrysotile.

According to the embodiment of the present invention, it is possible to detect EML4-ALK mutation using blood circulating cancer cells even in patients with lung cancer whose ALK-FISH test could not be performed due to difficulty in collecting lung cancer tissue, It is easy to confirm whether or not the anticancer drug is suitable for administration.

It should be understood that the effects of the present invention are not limited to the above effects and include all effects that can be deduced from the detailed description of the present invention or the configuration of the invention described in the claims.

FIG. 1 shows a process of separating blood circulating cancer cells.
2 is a photograph of blood circulating cancer cells separated by a high-density microchip.
FIG. 3 is a graph showing the growth and cleavage of blood circulating cancer cells cultured in the culture solution according to the present invention, and the growth and division of blood circulating cancer cells cultured in a general culture solution.
FIG. 4 is a graph showing the growth and cleavage of blood circulating cancer cells cultured in the hydrogel-coated culture plate used in the present invention and the growth and cleavage of blood circulating cancer cells cultured in a general culture plate using the culture solution according to the present invention Graph.
FIG. 5 shows the detection results of the V1 type gene mutation by the known method of detecting V1 type gene mutation of EML4-ALK.
FIG. 6 shows the detection results of the V1 type gene mutation using a primer for detecting V1 type gene mutation of EML4-ALK according to the present invention.
FIG. 7 shows detection results of V1 type gene mutation by nested PCR using a primer for detecting V1 type gene mutation of EML4-ALK according to the present invention.
FIG. 8 shows the detection result of the V3 gene mutation by the known method of detecting V3 type gene mutation of EML4-ALK.
FIG. 9 shows the detection results of the V3 type gene mutation using the primer for detecting the V3 type gene mutation of EML4-ALK according to the present invention.
FIG. 10 shows the results of detection of V3 type gene mutation by nested PCR using a primer for detecting V3 type gene mutation of EML4-ALK according to the present invention.
FIG. 11 shows the results of application to actual patient samples using a primer for detection of V1 type gene mutation of EML4-ALK according to the present invention.
Fig. 12 shows a qRT-PCR graph of the V1 type gene mutation of EML4-ALK in the H3122 cell line.
Fig. 13 shows a qRT-PCR graph of the V3 type gene mutation of EML4-ALK in the H2228 cell line.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described with reference to the accompanying drawings. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. In order to clearly illustrate the present invention, parts not related to the description are omitted, and similar parts are denoted by like reference characters throughout the specification.

Throughout the specification, when a part is referred to as being "connected" (connected, connected, coupled) with another part, it is not only the case where it is "directly connected" "Is included. Also, when an element is referred to as " comprising ", it means that it can include other elements, not excluding other elements unless specifically stated otherwise.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The singular expressions include plural expressions unless the context clearly dictates otherwise. In this specification, the terms "comprises" or "having" and the like refer to the presence of stated features, integers, steps, operations, elements, components, or combinations thereof, But do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, or combinations thereof.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings.

(A) obtaining a liquid biopsy sample from a cancer patient; (b) separating blood circulating cancer cells from the liquid biopsy sample using the biochip; and (c) (D) separating RNA from the separated blood circulating cancer cells and performing qRT-PCR using the separated RNA as a template and using two qRT-PCR primers; (e) And performing nested PCR using the two nested primers for the two qRT-PCR primers as a template, (f) detecting the mutation type of the EML-ALK gene as a product from the nested PCR .

The liquid biopsy refers to a test for collecting a gene fragment of cancer in a body fluid such as blood or plural, without invasive procedures such as puncture or incision. That is, the liquid biopsy can be used to make detailed observations on cancer development and metastasis by analyzing the cancer cell-derived DNA existing in the blood or ascites of the body part only by a body fluid test such as blood. According to an embodiment of the present invention, the sample for liquid biopsy may include blood, synovial fluid, ascites, pleural fluid, cerebrospinal fluid, and peritoneal fluid. According to a preferred embodiment of the present invention, the liquid biopsy sample may be blood.

The term circulating tumor cells (CTC) refers to tumor cells found in the peripheral blood of a malignant tumor patient. Circulating cancer cells are very rare and the amount of sample available is very limited. Techniques in the detection and characterization of circulating cancer cells include, but are not limited to, multiple reverse transcription-polymerase chain reaction methods, imaging-based approaches, microfiltration and microchip devices. Blood circulating cancer cells can act as tumor biologic markers that can inevitably provide individualized therapy and follow-up after treatment with liquid biopsy specimens. In addition, blood circulating cancer cells can be used as targets for understanding the biological characteristics of the tumor and the sowing of tumor cells, but are not limited thereto. According to an embodiment of the present invention, the blood circulating cancer cell may be a lung cancer-derived cell. According to a preferred embodiment of the present invention, the blood circulating cancer cells may be non-small cell lung cancer-derived cells.

The biochip refers to a solid substrate made of an inorganic material such as a semiconductor, which can be obtained by densely integrating and combining DNA, protein, enzyme, antibody, microorganism, animal or plant cell, Is a device or device that utilizes the inherent functions of biomolecules and obtains biological information such as gene expression pattern, gene binding, protein distribution, or biochemical process, reaction rate, or information processing speed.

The high density microchip refers to a biochip capable of separating a substance of a specific size based on the principle of operation of the biochip.

The high-density microchip is capable of capturing blood circulating cancer cells at a recovery rate of about 90% within 10 minutes based on the size difference of blood cells.

In an embodiment of the present invention, the pore size of the high-density microchip may be preferably 5.5 to 8.5 mu m, more preferably 6.5 to 7.5 mu m. When the pore size is less than 5.5 μm, the red blood cells and the white blood cells can not pass through the chip and are not removed because of the chip. When the pore size is larger than 8.5 μm, the pore size is smaller than the size of blood circulating cancer cells and immune cells The blood circulating cancer cells and the immune cells of the cursor can not pass through the chip and the blood circulating cancer cells and the immune cells can not be selectively recovered. In one embodiment of the present invention, the pores of the high-density microchip may be circular, square or elliptical, and preferably rectangular.

In one embodiment of the present invention, the pores of the high density microchip may be arranged in a regular pattern. In another embodiment of the present invention, the high-density microchip may be made of stainless steel, nickel, aluminum or copper. The pores may be formed by etching using MEMS (Micro-electro Mechanical Systems) technology.

The gap between two adjacent pores of the pores is formed to be narrower than the diameter of blood circulating cancer cells and immune cells. According to an embodiment of the present invention, the gap between the two pores may be formed to be 45 to 65% with respect to the diameter of blood circulating cancer cells and immune cells. The high-density microchip is not deformed by the pressure of blood or solutions flowing through the passages.

After the blood passes through the pores, it is discharged out, and blood circulating cancer cells in the blood do not pass through the pores and remain on the surface. According to an embodiment of the present invention, the blood circulating cancer cell may be a lung cancer-derived cell. According to a preferred embodiment of the present invention, the blood circulating cancer cells may be non-small cell lung cancer-derived cells.

Non-target cells, that is, red blood cells with higher strains than blood circulating cancer cells, readily pass through the pores.

Blood circulation After filtration of cancer cells, blood circulating cancer cells can supply the solution in a reverse or forward direction and discharge it out. According to one embodiment of the present invention, the solution can be supplied in the reverse direction so as to minimize the damage of blood circulating cancer cells. The solution may be supplied by a syringe, a syringe pump, a plunger pump, or the like. According to one embodiment of the present invention, the solution may consist of diluent, water and diluting acid to dilute the blood. Blood circulating cancer cells and immune cells which are discharged out of the solution by the supply of the solution can be easily collected by collecting in a container such as a test tube or a culture dish.

On the other hand, blood circulating cancer cells having a diameter of 7.5 to 15 탆 pass through a tube having a diameter of 8 탆 when a pressure of about 100 mmHg is applied. A blood circulating cancer cell having a diameter of 15 탆 passing through a tube having a diameter of 8 탆 has a strain of about 53%. Back wash, that is, the solution flows in the direction opposite to the blood flow, and when the blood circulating cancer cells having a diameter of 7.5 μm are discharged outside, the gap between the two pores is less than 4 μm considering the strain of blood circulating cancer cells desirable. For example, non-small cell lung cancer and breast cancer are known to have a diameter of about 40 μm in their cancer cells. Considering the strain of blood circulating cancer cells having a diameter of 40 m in backwashing, it is preferable to set the interval between the two pores to 21 mu m or less. When blood circulating cancer cells having a diameter of 7.5 mu m exist between two pores exceeding the gap of 4 mu m, blood circulating cancer cells may not be detached from the surface while being deformed by the solution flow. In addition, cancer cells with a diameter of 40 mu m may not fall off from the surface between two pores exceeding 21 mu m in spacing. In the high-density microchip according to the present invention, the gap between the two pores is 45 to 65% of the diameter of the circulating cancer cells in consideration of the pressure of the solution. Circulating cancer cells of 7.5 [mu] m in diameter may not be removed from the surface between the two pores by backwashing if the interval exceeds 65%, i.e., exceeds about 4.9 [mu] m. Blood circulating cancer cells of 40 m in diameter may not be separated from the surface between the two pores by backwashing when the interval exceeds 65%, that is, exceeds 26 m. When the solution pressure is increased by forcibly dropping blood circulating cancer cells stuck to the surface between the two pores, blood circulation cancer cells are damaged and the collection rate of living circulating cancer cells is lowered. When the distance to blood circulating cancer cells of 7.5 mu m in diameter is less than 45%, i.e., less than about 3.375 mu m, there is a high possibility that the high-density microchip is broken by the flow of blood and solution.

In one embodiment of the present invention, the high-density microchip allows the sample to pass repeatedly. Specifically, after blood circulation cancer cells are once separated from the high-density microchip, the separated blood circulation cancer cells can be once again loaded into the high-density microchip and separated, and the separation process can be repeated.

In one embodiment of the present invention, the blood circulation cancer cell separation using the high-density microchip is performed not by applying a specific artificial pressure after loading the solution containing the circulating cancer cells into the high-density microchip, Circulating cancer cells can be separated. The separation of blood circulating cancer cells through the high-density microchip according to the present invention can minimize the damage caused by artificial pressure on blood circulating cancer cells, thereby maintaining the state existing in the patient's body.

In one embodiment of the present invention, the high-density microchip is used to minimize the blood circulation cancer cell damage by the high-density microchip when blood circulating cancer cells are separated, or to make the high- Or may be coated with a specific material to make blood circulation cancer cell recovery rates more efficient. Specifically, the specific substance may be an antibody capable of specifically binding to blood circulating cancer cells, and may be a biomaterial that does not physically or chemically damage cells. According to one embodiment of the present invention, the specific substance may be BSA (Bovine Serum Albumin) or an antibody. The antibody may be composed of, for example, an anti-epithelial cell adhesion molecule antibody, an anti-EpCAM antibody, or an anti-CK antibody. According to a preferred embodiment of the present invention, the specific substance may be BSA (Bovine Serum Albumin).

The BSA (bovine serum albumin) solution refers to bovine serum albumin. The molecular weight of the protein is about 66.4 kDa, which is the most abundant protein in most animals. BSA can be added as a nutrient in cell culture in biochemistry / biology, and it is also used as a standard for obtaining a calibration curve in the quantification of proteins. When using restriction enzymes, it is necessary to use a small amount of enzyme (protein) It may be added to supplement the protein concentration in the solution. In addition, nonspecific binding, that is, nonspecific binding to an unwanted protein or an unwanted position of the antibody, can be detected in a number of biochemical experiments (Western blot, Immunocytochemistry, ELISA, etc.) It can also be used to prevent it.

According to an embodiment of the present invention, the peripheral blood may be reacted with the high-density microchip coated with the BSA solution using the centrifugal separation method to remove other biomolecules except the circulating cancer cells. According to an embodiment of the present invention, the separation using the high-density microchip may be performed by gravity, specifically, at an atmospheric pressure of 1000 to 1020 hPa. Preferably at atmospheric pressure of 1000 to 1015 hPa. More preferably at an atmospheric pressure of 1000 to 1013 hPa.

According to one embodiment of the present invention, the BSA solution may be coated on the upper surface, the lower surface, or the inner surface of the pores of the high-density microchip. Preferably on both the upper and lower surfaces of the high-density microchip and the inner surfaces of the pores

According to one embodiment of the present invention, the BSA solution coating may be in a concentration of 0.05 to 0.15%. According to a preferred embodiment of the present invention, the BSA solution coating may have a concentration of 0.08 to 0.012%.

According to one embodiment of the present invention, the BSA solution coating may be treated for 5 to 15 minutes. According to one preferred embodiment of the present invention, the BSA solution coating can be treated for 8 to 12 minutes.

Referring to FIG. 1, in order to remove the blood cells from the patient's blood, the antibody polymer is added to the blood of the collected patient, followed by mixing well and then reacting at room temperature. Then, PBS solution containing 1% FBS was added, and Ficoll solution was added to the reaction solution, and then the blood cells were firstly removed by centrifugation. In the present invention, unnecessary blood cells are firstly removed, and the high-density microchips coated with the BSA solution are used to filter the red blood cells, thereby isolating highly pure circulating cancer cells. Separated circulating cancer cells are identified through staining. When the high-density microchip coated with BSA is used, it is possible to isolate the blood circulating cancer cell damage to be separated in the present invention in a minimized state. Thus, the high-purity blood circulating cancer cell can be obtained by the EML4-ALK gene mutation analysis method of the present invention It can significantly increase its efficiency to work effectively.

According to an embodiment of the present invention, after the step of separating blood circulating cancer cells from the liquid biopsy sample using the biochip, a step of short-term culturing the separated blood circulating cancer cells may be further performed.

In one embodiment of the present invention, the culture medium used in the short-term culture may be at least three selected from the group consisting of insulin, transferrin, Epidermal Growth Factor (EGF) and ROCK (Rho Kinase) inhibitor.

The insulin is one of the hormones of the human metabolism system and is secreted from the Langerhans island beta cells of the organs and serves to keep the blood glucose level in the blood constant. When the blood sugar level is higher than a certain level, insulin is secreted, and the glucose in the blood is introduced into the cells to accelerate the action of storing it in the form of polysaccharide (glycogen) again. The culture medium used in the short-term culture step according to the present invention can promote the cell growth and cleavage more than the conventional serum-free tumor cell culture medium in culturing blood cancer cells by including the insulin. According to one embodiment of the present invention, the insulin is administered at a dose of 3 to 50 ng / ml, 3 to 45 ng / ml, 3 to 40 ng / ml, 3 to 30 ng / ml, 4-50 ng / To 30 ng / ml, 5 to 50 ng / ml, 5 to 45 ng / ml or 5 to 30 ng / ml.

The transferrin is a type of? Globulin, which is an iron transport protein that binds to two molecules of trivalent iron ions absorbed in the serum to supply iron necessary for cell proliferation or hemoglobin production via a transferrin receptor. More than 99% of the iron in the serum binds to transferrin and normally about one-third of the transferrin can bind to iron. The culture medium used in the short-term culture step according to the present invention can further promote cell growth and division in the blood cultured cancer cells by including the transferrin. According to an embodiment of the present invention, the transferrin is administered at a dose of 3-50 ng / ml, 3-45 ng / ml, 3-40 ng / ml, 3-30 ng / ml, 4-50 ng / ml, 4-45 ng / ml , 4 to 30 ng / ml, 5 to 50 ng / ml, 5 to 45 ng / ml or 5 to 30 ng / ml.

The Epidermal Growth Factor (EGF) is a polypeptide growth factor that binds to epidermal growth factor receptor and promotes cell growth and division. In addition to promoting protein synthesis and RNA synthesis, the epithelial growth factor may induce ornithin decarboxylase. The culture solution used in the short-term culture step according to the present invention can further promote cell growth and division in the blood cultured cancer cells by including the epithelial growth factor as compared with the conventional blood-like cells culture solution. According to an embodiment of the present invention, the epithelial growth factor is administered at a dose of 0.5 to 10 ng / ml, 0.5 to 9 ng / ml, 0.5 to 8 ng / ml, 0.5 to 7 ng / ml, 0.5 to 6 ng / 0.7 to 10 ng / ml, 0.7 to 9 ng / ml, 0.7 to 8 ng / ml, 0.7 to 7 ng / ml, 0.7 to 6 ng / ml, 8 ng / ml, 1 ~ 7 ng / ml, 1 ~ 6 ng / ml or 1 ~ 5 ng / ml.

The Rho-associated protein kinase (ROCK) inhibitor refers to a compound that targets or inhibits the function of Rho kinase (ROCK). Here, Rho kinase is a kinase belonging to the AGC family of serine-threonine kinases (PKA / PKG / PKC). The Rho kinase is involved in the process of controlling the movement and morphology of cells by acting on the cytoskeleton. Specifically, the Rho kinase may act as a regulator of cell migration and actin organization. The Rho kinase is associated with neurodegenerative diseases such as diabetes, hemorrhagic cerebrovascular disease, Parkinson ' s disease, and the Rho kinase inhibitor can be used for the treatment and inhibition of the Rho kinase related diseases. According to one embodiment of the present invention, the ROCK inhibitor may be at least one selected from the group consisting of Fasudil, Ripasudil, RKI-1447 and Y27632. The fasudil is one of the ROCK inhibitors and can be used for the treatment of cerebral vasospasm and can also be effective in the treatment of pulmonary hypertension. The rifaidalil may act as a ROCK inhibitor as a derivative of fasudil and may be used for the treatment of glaucoma or ocular hypertension. The RKI-1447 can inhibit ROCK1 and ROCK2. The Y27632 can inhibit ROCK1 and ROCK2 by competing with ATP to bind the catalytic site of the enzyme through the cell. According to an embodiment of the present invention, the ROCK inhibitor may be administered at a dose of 3 to 30 μM, 3 to 27 μM, 3 to 24 μM, 3 to 21 μM, 4 to 20 μM, 4 to 30 μM, 4 to 27 μM, 4 to 24 μM, 5 to 30 μM, 5 to 27 μM, 5 to 24 μM, 5 to 21 μM or 5 to 20 μM.

The culture plate used in the short-term culture according to the present invention may have a surface that prevents cell adhesion. According to an embodiment of the present invention, blood cancer cells can be cultured in a floating state, so that the surface of the culture plate can be coated to prevent cell adhesion. The surface coating of the culture plate may consist of a hydrogel. The hydrogel refers to a gel containing water as a dispersion medium, and the hydrosol loses its fluidity due to cooling, or the hydrophilic polymer having a three-dimensional network structure and a microcrystalline structure is formed by swelling with water. Specifically, the hydrogel is a hydrophilic polymer crosslinked by a cohesive force such as a covalent bond, a hydrogen bond, a van der Waals bond, a physical bond or the like. The hydrogel is a three-dimensional polymer network capable of swelling a large amount of water contained therein Structure. Such water absorption shows similar behavior to the tissue of the living body. The hydrogel may undergo phase transition by temperature, pH, etc., and the swelling ratio may be discontinuously changed, and may be used for contact lenses, medical electrodes, and cell cultures. According to an embodiment of the present invention, the hydrogel may be covalently coated on the culture plate to prevent blood cancer cells from adhering to the surface of the culture plate. According to another embodiment of the present invention, the hydrogel may have a hydrophilic property while having a neutral charge. Having the neutral charge means that the charge has a state of neither positive nor negative, and the hydrophilicity means a strong affinity for water, which means that it can be dissolved well in water.

In one embodiment of the present invention, the short-term culturing step may be performed for 1 to 15 days from the start of culturing. In another embodiment of the present invention may be from 5 to 15 days, and preferably from 10 to 15 days. And still more preferably 12 to 15 days.

According to one embodiment of the present invention, the ALK-positive non-small cell lung cancer is lung cancer caused by the EML4-ALK mutation by fusion of two genes, ALK and EML4, and the EML4-ALK mutation is the exon and / Or depending on the intron region, have various mutation types as shown below.

Variant 1: exon 1-13 (EML4) + exon 20-29 (ALK)

Variant 2: exon 1-20 (EML4) + exon 20-29 (ALK)

Variant 3a: exon 1-6a (EML4) + exon 20-29 (ALK)

Variant 3b: exon 1-6b (EML4) + exon 20-29 (ALK)

Variant 4a: exon 15 (EML4) + exon 20-29 (ALK)

Variant 4b: Linker consisting of exon 14 (EML4) + 11bp size oligonucleotide + exon 20-29 CALK)

Variant 5a: exon 2 (EML4) + exon 20-29 (ALK)

Variant 5b: exon 2 (EML4) + intron 19 (ALK) + exon 20-29 (ALK)

Among these mutations, there were two types of mutations in Korea (Variant 1, V1) and Mutant 3 (Variant 3, V3). In 167 patients with non-small cell lung cancer, 10 patients had EML4-ALK fusion mutations When confirmed, there were reports that four of them were V1 type mutations and two were V3b type mutations ( J. Korean Med. Sci., 27: 228-230, 2012 ).

There are two isoforms in the V3 type, and 33bp in the EML4 Intron 6 in the V3b type. Therefore, two types of PCR products can be generated when detecting V3 type PCR.

According to an embodiment of the present invention, the mutation type of the EML4-ALK gene may be V1 type, V2 type, V3a type, V3b type, V4a type, V4b type, V5a type or V5b type, Or V3 type.

The step of isolating the RNA may be performed according to a method commonly used in the art, and may be carried out using a commercially available RNA extraction kit. Also, in the step of performing qRT-PCR, qRT-PCR uses separated RNA as a template and uses as primers for reverse qRT-PCR of two qRT-PCR primers of the present invention as primers, and reverse transcriptase Through the reaction, a complementary DNA strand can be synthesized and then a PCR reaction can be performed. The PCR reaction can be carried out using a PCR reaction mixture containing various components known in the art necessary for the PCR reaction. The PCR reaction mixture includes an appropriate amount of DNA polymerase, dNTP, PCR buffer solution and distilled water (dH ₂ O) in addition to the complementary DNA synthesized by the reverse transcription reaction and the qRT-PCR primers provided in the present invention. The PCR buffer comprises Tris -HCl (Tris-HCl), MgCl 2, KCl and the like. At this time, MgCl ₂ concentration greatly affects amplification specificity and yield. In general, when Mg ²⁺ is excessive, nonspecific PCR amplification products are increased, and when Mg ²⁺ is insufficient, the yield of PCR products is decreased. The PCR buffer solution may further contain an appropriate amount of Triton X-100 (Triton X-100). PCR also involves denaturation of the DNA followed by denaturation; Annealing; Followed by a cycle of amplification and extension followed by final elongation at 72 ° C. In the above, denaturation and amplification can be performed at 94 to 95 ° C and 72 ° C, respectively, and the temperature at the time of binding can be varied depending on the type of the primer. According to one embodiment of the present invention, the primer for qRT-PCR of the present invention can be performed at 55 to 64 ° C. The time and number of cycles in each step can be determined according to the conditions commonly practiced in the art.

The detection limit of the known primers that have been used for the detection of EML4-ALK mutation is 0.1 to 1%. When the sample is in a cancerous tissue, there is no problem in detecting the mutation. However, in order to use a method of isolating and detecting circulating cancer cells Should be confirmed at 0.1% or less. Therefore, a primer having a low detection limit is required. In the present invention, in order to design a primer having high accuracy and detection efficiency, a primer was designed by the following method. The design of the primer is determined by using the primer3 (http: //bioinfo.ut.ee/orimer3-0.4.O/) primer design program, and the NCBI homepage (htto: //www.ncbi.nlm.nih. gov / blasted to avoid the possibility of primer binding to other parts of the EML4-ALK site and the possibility of hairpin structure, and to avoid direct dimer formation by directly comparing the homology between primer pairs, and GC The PCR products were designed so that the primers can be used in combination with any combination of primers in a manner similar to the Tm values of the four primers. Avoiding repeated or sequential sequences, and using cDNA as a template, was designed between possible exon junctions to prevent multiple bands from binding to the genomic DNA.

According to another embodiment of the present invention, one of the two primers for qRT-PCR is one primer for qRT-PCR selected from the group consisting of primers represented by SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 4 And the other is a primer for reverse qRT-PCR shown in SEQ ID NO: 2.

According to an embodiment of the present invention, the two primers for qRT-PCR include a pair of primers represented by SEQ ID NO: 1 and SEQ ID NO: 2; A pair of primers represented by SEQ ID NO: 3 and SEQ ID NO: 2; And a pair of primers represented by SEQ ID NO: 4 and SEQ ID NO: 2.

According to another embodiment of the present invention, the two primers for qRT-PCR may be used for detecting the V1 type gene mutation of EML4-ALK and the primer pair represented by SEQ ID NO: 1 and SEQ ID NO: 2, And a primer pair represented by SEQ ID NO: 2; And the primer pair represented by SEQ ID NO: 3 and SEQ ID NO: 2 can be used to detect V3 type gene mutation of EML4-ALK.

In the nested PCR, the DNA of the PCR product can be isolated according to a method well known in the art. Preferably by agarose gel or polyacrylamide gel electrophoresis or fluorescence analysis (ABI prism 3100 genetic analyzer-electropherogram). At this time, in order to use the fluorescence analyzer, PCR is performed in the step of performing qRT-PCR using two qRT-PCR primers attached with fluorescent dyes known in the art.

The addition of the nested PCR step minimizes errors due to nonspecific reactions when detecting PCV, which can be useful when needed.

The PCR amplification result can be confirmed by agarose gel electrophoresis. After electrophoresis, electrophoresis results can be analyzed by ethidium bromide staining. Methods for performing general reverse transcription, PCR and analysis of the results are well known in the art.

According to one embodiment of the present invention, one of the two nested primers may be a forward nested primer and the other may be a reverse nested primer.

According to another embodiment of the present invention, one of the two nested primers is a forward nested primer selected from the group consisting of primers represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 10 And the other is a reverse nested primer selected from the group consisting of the primers represented by SEQ ID NO: 2 or SEQ ID NO: 9, but any pair of primers capable of amplifying the qRT-PCR product can be used without limitation .

According to another embodiment of the present invention, the two nested primers include a pair of primers represented by SEQ ID NO: 1 and SEQ ID NO: 9; And the primer pair shown in SEQ ID NO: 10 and SEQ ID NO: 2 can be used to detect the V1 type gene mutation of EML4-ALK, and the pair of primers shown in SEQ ID NO: 3 and SEQ ID NO: 2; And a primer pair represented by the primer pair shown in SEQ ID NO: 4 and SEQ ID NO: 2 can be used to detect the V3 type gene mutation of EML4-ALK.

The standard recombinant DNA and molecular cloning techniques used in the present invention are well known in the art and are described in the following references (Sambrook, J., Fritsch, EF and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989); by Silhavy, TJ, Bennan, ML and Enquist, LW, Experiments with Gene Fusions, Cold Spring Harbor Laboratory: Cold Spring Harbor, by Ausubel, FM et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-Interscience (1987)).

A cancer patient screening method according to another aspect of the present invention is characterized in that the mutation obtained by the EML4-ALK gene mutation analysis method

(i) exons 1-13 of the EML4 gene and exons 20-29 of the ALK gene;

(ii) exons 1-20 of the EML4 gene; exons 20-29 of the ALK gene;

(iii) exons 1-6a of the EML4 gene and exons 20-29 of the ALK gene;

(iv) exons 20-29 of the exon 1-6b + ALK gene of the EML4 gene;

(v) exon 15 of the EML4 gene and exon 20-29 of the ALK gene;

(vi) a linker consisting of an exon 14 + 11bp-sized oligonucleotide of the EML4 gene and an exon 20-29 of the ALK gene;

(vii) exon 2 of the EML4 gene + exon 20-29 of the ALK gene; And

(viii) Exon 2 of the EML4 gene, intron 19 of the ALK gene, and exon 20-29 of the ALK gene are consistent with one selected from the group consisting of:

According to one embodiment of the present invention, the cancer may be lung cancer, preferably non-small cell lung cancer.

According to another embodiment of the present invention, the anticancer agent may be chrysotile.

The target anticancer drug for non-small cell lung cancer caused by EML4-ALK mutation is cryotorib (XALKORI, Pfizer). Currently, the treatment of non-small cell lung cancer is the Iressa / / It is proceeding in a way to select the treatment modality among conventional cytotoxic anticancer drugs.

However, in patients unable to invasive biopsy of lung cancer tissue, genetic testing for the EML4-ALK fusion mutation could not be performed and the efficacy of administration of clozotanib could not be confirmed. There was no.

The method of screening cancer patients according to the present invention comprises isolating blood circulating cancer cells from the blood of non-small cell lung cancer patients and confirming the presence of the EML4-ALK fusion mutation of the blood circulating cancer cells, Can be determined.

Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily carry out the present invention. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

Example 1: High-density microchip fabrication

The high-density microchip used in the experiment has a pore-size of 5.5 to 8.5 μm, and a white blood cell (WBC) and a red blood cell (RBC) smaller than 5.5 μm It is a microchip that is designed to remove by passing a chip, and target cells larger than 5.5 μm in size to be picked up on the chip to selectively recover a specific size.

In order to confirm the cell recovery rate of the high-density microchip, experiments were conducted by spiking 10, 100, and 1000 cancer cells in a cancer patient and passing the same through a chip to see the recovery rate of cancer cells on the chip . The results are shown in Table 1 below.

sample Spiked cell number Number of cells recovered Cell recovery (%) One 10 9 90 2 100 86 86 3 1000 850 85

As a result of counting the cell recovery rate of the chip, the cell recovery rate was about 80% or higher. In order to confirm that the recovered cells are cancer cells, the CK antibody used in the cancer assay was stained. Was confirmed to be a cancer cell.

Example 2: Separation of blood circulating cancer cells (CTC)

1. Add 250 μl of antibody polymer to 5 ml of blood, mix for 3 seconds, and incubate at room temperature for 20 minutes.

2. Add 5 ml of PBS containing 1% FBS.

3. Carefully raise 10 ml of the reaction solution in a 50 ml tube containing 15 ml of Ficoll solution.

4. Centrifuge the solution at 1200 g for 20 minutes to remove blood cells first.

5. In order to prevent unnecessary cell adsorption, high density microchip (HD Microporous chip) is treated with 0.1% BSA solution for 10 minutes and then rinsed with PBS.

6. Ficoll's upper layer solution is placed on the filter net and the remaining red blood cells are gravity filtered to isolate highly purified blood circulating cancer cells. This prevents blood circulation cancer cells from being damaged by not performing treatment such as centrifugation or immunobead.

7. Isolated blood circulating cancer cells are identified through staining.

Example 3. Short-term culture of isolated blood circulating cancer cells

The circulating cancer cells isolated by the high density microchip according to the present invention were seeded on a Ultra low attachment culture plate coated with a hydrophilic hydrogel with neutral charge. The culture plate contained a culture medium containing 11ng / ml of insulin, 22ng / ml of transferrin, 2ng / ml of EGF and 8 占 R of ROCK inhibitor. The short-term culture was carried out for 14 days in a cell culture incubator for 37 days Lt; 0 > C and 5 to 10% CO ₂ .

Example 4. Identification of blood circulating cancer cells

In order to confirm that the blood circulating cancer cells cultured in the short-term according to Example 3 are cancer cells through the staining method, the cell staining process is performed using the following method.

1. Cytospin, a cell centrifugation process, is performed to fix the recovered cells to a staining slide.

2. Perform permeabilization to allow the antibody to enter the cell.

3. Perform a washing process with PBS.

4. Perform 1% BSA (bovine serum albumin) using PBS and perform a blocking process to reduce non-specific binding and endogenous peroxidase activity.

5. EpCAM (epithelial cell adhesion molecule), CK (cytokeratin) and CD (cluster of differentiation) 45 are reacted as a primary antibody at room temperature for 60 minutes.

6. The fluorescently labeled secondary antibody that binds to the primary antibody is reacted at room temperature for 60 minutes.

7. Perform a washing process with PBS.

8. Finally, to stain the cell nuclei, add DAPI (4 ', 6-diamidino-2-phenylindole) solution and cover glass cover for 10 minutes at room temperature.

9. While observing the stained cells, calculate the stained rate and recovery rate manually.

Comparative Example 1. Changes in the number of cultured cells in circulating cancer cells according to the culture medium

In general, the degree of cleavage and growth of blood circulating cancer cells in the cell culture medium used for cell culture and the culture medium according to the present invention was compared. The culture media used for cell culture were 25 nM sodium selenite, 50 nM hydrocortisone, 0.01 mM ethanolamine, 0.01 mM phosphorylethanolamine, 100 pM triiodothyronine, 0.5% (w / v) bovine serum albumin, 10 mM HEPES, 0.5 mM sodium pyruvate, 4.5 mM L-glutamine, and 1X antibiotic-antimycotic. The culture solution according to the present invention is the same as that of the third embodiment. The culture conditions were the same as in Example 3 except that a general plate was used.

In FIG. 3, CD45- is a biomarker for blood circulating cancer cells to be cultured. Normal growth medium means a cell culture medium, CG growth medium means a culture medium according to the present invention. FIG. 3 shows that blood circulating cancer cells are cultured more in the culture solution according to the present invention, which means that the culture solution according to the present invention is more effective in the cleavage and culture of blood circulating cancer cells than a general culture solution.

Comparative Example 2. Changes in the Number of Cultured Cells of Circulating Cancer Cells in Cultured Plates Coated with Hydrogel

In the situation where the culture solution according to the present invention was used, a comparative culture with a general culture plate was carried out to measure the effect of the hydrogel-coated culture plate on the cell growth and cleavage of blood circulating cancer cells. The culture solution according to the present invention is the same as in the third embodiment.

Referring to FIG. 4, the normal culture type shows a general cell culture plate (cells attached to the culture plate surface), and the low attachment type is the culture plate coated with the hydrogel used in the present invention. FIG. 4 shows that the blood circulating cancer cells cultured on the hydrogel-cured culture plate were further cultured using the culture solution according to the present invention, and that the culture on the hydrogel-coated culture plate was used for the cleavage and culture of blood circulating cancer cells Which is more effective.

Example 5: Detection of V1 type mutation of EML4-ALK using qRT-PCR

The H3122 cell line (Lane-Meyer's Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02115), a lung cancer cell line with a variant of V1 type of EML4-ALK, was cultured and RNA was extracted. CDNA was synthesized from the extracted RNA, and the synthesized cDNA was sequentially diluted and used as a template for qRT-PCR for V1 type detection of EML4-ALK.

PBMC cells and A549 cells (ATCC, CCL-185) were used as negative controls. The composition of the mixture for the qRT-PCR reaction is shown in Table 2.

Mixture composition for PCR reaction matter content 2X SYBR Green 10 μl Forward Primer 1 μl Reverse Primer 1 μl Template 1 μl Distilled water 7 μl Total volume 20 쨉 l

The PCR cycle was performed at 95 ° C for 10 minutes, followed by 40 cycles of 95 ° C for 30 seconds, 60 ° C for 30 seconds, and 72 ° C for 40 seconds as one cycle, and extension at 72 ° C for 10 minutes

The PCR primers used are as follows.

EML4 (E12) -FP: 5'-CACACCTGGGAAAGGACCTA-3 '(V1 forward primer)

No. 1)

ALK (E20) -RP: 5'-ACTACTGCTTTGCTGGCAAGACCT-3 '(V1 reverse primer) (SEQ ID NO: 2)

Control primer sequence (FP): 5'-GTGCAGTGTTTAGCATTCTTGGGG-3 '(forward

primer) (SEQ ID NO: 5)

Control primer sequence (RP): 5'-ATCCAGTTCGTCCTGTTCAGAGC-3 '(reverse

primer) (SEQ ID NO: 6)

The amplified qRT-PCR product was electrophoresed on an agarose gel to identify bands, and the identified bands were separated and sequenced.

As a result, the V1 mutation could be detected at a detection limit of 0.1% to 1% in both the PCR method using the known primers (Fig. 5) and the qRT-PCR method using the primer of the present invention (Fig. 6) A more clean band could be identified in the method of the example. Here, 0.1% means that one circulating cancer cell per 1,000 white blood cells can be detected.

Example 6: Detection of V3 type mutation of EML4-ALK using qRT-PCR

The H2228 cell line (ATCC, CRL-5935), a lung cancer cell line having the V3 type mutation of EML4-ALK, was cultured and RNA was extracted. CDNA was synthesized from the extracted RNA, and the synthesized cDNA was sequentially diluted and used as a template for qRT-PCR for V3 type detection of EML4-ALK. QRT-PCR was performed in the same manner as in Example 5 except for the primers used.

When performing qRT-PCR using the EML4 (E1E2) -FP primer (SEQ ID NO: 3) and ALK (E20) -RP primer (SEQ ID NO: 2), PCR products of 789 bp (V3a type) or 822 bp (V3b type) (V3a type) or 420bp (V3b) when qRT-PCR is performed using the EML4 (E4) -FP primer (SEQ ID NO: 4) and the ALK (E20) -RP primer Type) qRT-PCR product is amplified.

The qRT-PCR primers used are as follows.

EML4 (E1E2) -FP: 5'-CCAAAACTGCAGACAAGCAT-3 '(V3 forward primer) (SEQ ID NO: 3)

EML4 (E4) -FP: 5'-CACAAATTCGAGCATCACCTTCTC-3 '(V3 forward primer) (SEQ ID NO: 4)

ALK (E20) -RP: 5'-ACTACTGCTTTGCTGGCAAGACCT-3 '(V3 reverse primer) (SEQ ID NO: 2)

Control primer sequence (FP): 5'-GTCAGCTCITGAGTCACGAGIT-3 '(forward primer) (SEQ ID NO: 7)

Control primer sequence (RP): 5'-ATCCAGITCGTCCTGITCAGAGC-3 '(reverse primer) (SEQ ID NO: 8)

The amplified qRT-PCR product was electrophoresed on an agarose gel to identify bands, and the identified bands were separated and sequenced.

As a result, the V3 mutation could be detected at a detection limit of 1% to 10% in the PCR method using known primers (FIG. 8), and the PCR method using the primer of the present invention (FIG. 9) E1E2) -FP primer, the detection limit of 1 to 10% was exhibited. When the EML4 (E4) -FP primer was used, the detection limit was 0.1 to 1%.

Example 7: Detection of V1 and V3 type mutations using nested PCR

In the Nested PCR for detecting mutations V1, 1 ^st PCR was EML4 (E12) -FP (standing column number 1) and 3078RR primer (SEQ ID NO: 9) were used, 2 ^nd PCR is RT-S fusion primer (SEQ ID NO: 10) and ALK (E20) -RP (SEQ ID NO: 2) were used.

3078RR primer sequence: 5'-ATCCAGTTCGTCCTGTTCAGAGC -3 '(SEQ ID NO: 9)

Fusion RT-S primer sequence: 5'-GTGCAGTGTTTAGCATTCTTGGGG-3 '(SEQ ID NO: 10)

As a result, as shown in FIG. 7, it was confirmed that in the case of the V1 type mutation, the mutation type could be detected at the detection limit of 0.001%, which is 1000 times more improved than the known technology,

EML4 (E1E2) -FP (SEQ ID NO: 3) and ALK (E20) -RP (SEQ ID NO: 2) were used for 1st PCR and EML4 (E4) -FP SEQ ID NO: 4) and ALK (E20) -RP (SEQ ID NO: 2) were used.

As a result, as shown in Fig. 10, it was confirmed that in the case of the V3 type mutation, the detection of the mutation type was possible with the detection limit of 0.1%, and the sensitivity was improved by 10 times.

Example 8 EMI.A-ALK Mutation Detection in Clinical Samples Using Nested PCR

The PCR and Nested PCR methods of Example 6 and Example 7 were used to detect EML4-ALK mutations in clinical samples.

Among the histologically proven non-small cell lung cancer patients, blood circulating cancer cells isolated from the blood of patients with EML4-ALK positive (IRB 1209-029-424, September 12, 2012) RNA was extracted. CDNA was synthesized from the extracted RNA, and nested PCR was performed by the method of Example 7 using the synthesized cDNA as a template.

As a result, as shown in FIG. 11, the detection of the V1 type mutation was confirmed, and it was confirmed that the EML4-ALK mutation could be detected in blood cancer cells of patients having the actual EML4-ALK mutation.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive. For example, each component described as a single entity may be distributed and implemented, and components described as being distributed may also be implemented in a combined form.

The scope of the present invention is defined by the appended claims, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included within the scope of the present invention.

<110> Cytogen, Inc. <120> A METHOD FOR ANALYZING EML4-ALK GENE VARIANCE <130> P20170105KR <160> 10 <170> KoPatentin 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> EML4 (E12) -Foward Primer Sequence <400> 1 cacacctggg aaaggaccta 20 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> ALK (E20) -Reverse Primer Sequence <400> 2 actactgctt tgctggcaag acct 24 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> EML4 (E1E2) -Foward Primer Sequence <400> 3 ccaaaactgc agacaagcat 20 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> EML4 (E4) -Foward Primer Sequence <400> 4 cacaaattcg agcatcacct tctc 24 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Control Foward Primer Sequence <400> 5 gtgcagtgtt tagcattctt gggg 24 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Control Reverse Primer Sequence <400> 6 gtgcagtgtt tagcattctt gggg 24 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Control Forward Primer Sequence <400> 7 gtcagctctt gagtcacgag tt 22 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Control Reverse Primer Sequence <400> 8 atccagttcg tcctgttcag agc 23 <210> 9 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> 3078RR Primer Sequence <400> 9 atccagttcg tcctgttcag agc 23 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Fusion RT-S Primer Sequence <400> 10 gtgcagtgtt tagcattctt gggg 24

Claims (19)

Obtaining a liquid biopsy sample from a cancer patient;
Separating blood circulating cancer cells from the liquid biopsy sample using the biochip;
Separating the RNA from the isolated blood circulating cancer cells;
Performing qRT-PCR using the separated RNA as a template and using two qRT-PCR primers;
Performing nested PCR using the product from the qRT-PCR as a template and using two nested primers for the two qRT-PCR primers; And
Detecting a mutation type of the EML-ALK gene as a product from the nested PCR;
Wherein the EML4-ALK gene mutation comprises the amino acid sequence of SEQ ID NO: The method according to claim 1,
Wherein the liquid biopsy sample is blood. The method according to claim 1,
Wherein the cancer is lung cancer. The method according to claim 1,
Wherein said cancer is non-small cell lung cancer. The method according to claim 1,
Wherein the step of isolating the blood circulating cancer cells is performed under an atmospheric pressure of 1000 to 1020 hPa. The method according to claim 1,
Wherein the biochip is a high-density microchip coated with a BSA solution. The method according to claim 6,
Wherein the high-density microchip has size specificity. The method according to claim 6,
Wherein the BSA solution coating is coated at a concentration of 0.05 to 0.15%. The method according to claim 1,
Wherein the mutation type of the EML4-ALK gene is a V1 type or a V3 type. The method according to claim 1,
Wherein one of the two qRT-PCR primers is a forward qRT-PCR primer and the other is a reverse qRT-PCR primer. 11. The method of claim 10,
Wherein the forward primer for qRT-PCR is one selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 4. 11. The method of claim 10,
Wherein the reverse qRT-PCR primer is SEQ ID NO: 2. The method according to claim 1,
Wherein one of the two nested primers is a forward nested primer and the other is a reverse nested primer. 14. The method of claim 13,
Wherein the forward nested primer is one selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 10. 14. The method of claim 13,
Wherein the reverse nested primer is SEQ ID NO: 2 or SEQ ID NO: 9. The mutation of the cancer patient obtained through the EML4-ALK gene mutation analysis method according to claim 1
(i) exons 1-13 of the EML4 gene and exons 20-29 of the ALK gene;
(ii) exons 1-20 of the EML4 gene; exons 20-29 of the ALK gene;
(iii) exons 1-6a of the EML4 gene and exons 20-29 of the ALK gene;
(iv) exons 20-29 of the exon 1-6b + ALK gene of the EML4 gene;
(v) exon 15 of the EML4 gene and exon 20-29 of the ALK gene;
(vi) a linker consisting of an exon 14 + 11bp-sized oligonucleotide of the EML4 gene and an exon 20-29 of the ALK gene;
(vii) exon 2 of the EML4 gene + exon 20-29 of the ALK gene; And
(viii) a method of screening cancer patients showing that the anticancer agent is potentially effective against the cancer patient if the EML4 gene is identical to the exon 2 + ALK gene intron 19 + the ALK gene exon 20-29. 17. The method of claim 16,
Wherein the cancer is lung cancer. 17. The method of claim 16,
Wherein the cancer is non-small cell lung cancer. 17. The method of claim 16,
Wherein the anticancer agent is chrysotriptib.

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